Tyler S. Quist

The impact of osteoblastic differentiation on osteosarcomagenesis in the mouse

Osteosarcomas remain an enigmatic group of malignancies that share in common the presence of transformed cells producing osteoid matrix, even if these cells comprise a minority of the tumor volume. The differentiation state of osteosarcomas has therefore become a topic of interest and challenge to those who study this disease. In order to test how the cell of origin contributes to the final state of differentiation in the transformed cells, we compared the relative tumorigenicity of Cre-LoxP conditional disruption of the cell cycle checkpoint tumor-suppressor genes Trp53 and Rb1 using Prx1-Cre, Collagen-1α1-Cre and Osteocalcin-Cre to transform undifferentiated mesenchyme, preosteoblasts and mature osteoblasts, respectively. The Prx1 and Col1α1 lineages developed tumors with nearly complete penetrance, as anticipated. Osteosarcomas also developed in 44% of Oc-Cre;Rb1fl/fl;Trp53fl/fl mice. We confirmed using 5-ethynyl-2′-deoxyuridine click chemistry that the Oc-Cre lineage includes very few actively cycling cells. By assessing radiographic mineralization and histological osteoid production, the differentiation state of tumors did not correlate with the differentiation state of the lineage of origin. Some of the osteocalcin-lineage-derived osteosarcomas were among the least osteoblastic. Osteocalcin immunohistochemistry in tumors correlated well with the expression of DNA methyl transferases, suggesting that silencing of these epigenetic regulators may influence the final differentiation state of an osteosarcoma. Transformation of differentiated, minimally proliferative osteoblasts is possible but may require such an epigenetic reprogramming that the tumors no longer resemble their differentiated origins.

Hearing preservation after middle fossa vestibular schwannoma removal: are the results durable?

Objective To describe 5-year hearing preservation rates following microsurgical excision of vestibular schwannoma (VS) via the middle cranial fossa (MCF) approach. Study Design Case series with chart review. Setting This study was performed at a tertiary care academic medical center. Subjects and Methods Fifty-seven subjects with VS underwent resection via an MCF approach between February 1998 and January 2009. Pure-tone average (PTA) and word recognition score (WRS) were obtained preoperatively, immediately postoperatively, and at 5-year follow-up. Results Preoperative serviceable hearing (American Academy of Otolaryngology—Head and Neck Surgery class A/B) was present in 49 (86%) of the 57 patients, with an average PTA of 23 dB (range, 1-50 dB) and an average WRS of 97% (range, 76%-100%). Immediate postoperative serviceable hearing was maintained in 27 (55%) patients, with an average PTA and WRS of 31 dB (5-50 dB) and 96% (70%-100%), respectively. Five-year follow-up was available for 16 of the 27 patients. Twelve (75%) of the 16 patients maintained serviceable hearing with an average PTA and WRS of 35 dB (4-49 dB) and 95% (84%-100%), respectively. Of the 16 subjects who did maintain class A or B hearing, the mean change in PTA and WRS was 5 dB and 0.4%, respectively. Of the 4 patients who did not maintain class A/B hearing, average change in PTA and WRS was 16 dB (4.5-23 dB) and 16% (0%-40%), respectively. Conclusions For patients with VS in whom serviceable hearing is preserved following the MCF approach, the long-term hearing outcome remains durable in most patients.

Zebrafish foxP2 zinc finger nuclease mutant has normal axon pathfinding.

foxP2, a forkhead-domain transcription factor, is critical for speech and language development in humans, but its role in the establishment of CNS connectivity is unclear. While in vitro studies have identified axon guidance molecules as targets of foxP2 regulation, and cell culture assays suggest a role for foxP2 in neurite outgrowth, in vivo studies have been lacking regarding a role for foxP2 in axon pathfinding. We used a modified zinc finger nuclease methodology to generate mutations in the zebrafish foxP2 gene. Using PCR-based high resolution melt curve analysis (HRMA) of G0 founder animals, we screened and identified three mutants carrying nonsense mutations in the 2nd coding exon: a 17 base-pair (bp) deletion, an 8bp deletion, and a 4bp insertion. Sequence analysis of cDNA confirmed that these were frameshift mutations with predicted early protein truncations. Homozygous mutant fish were viable and fertile, with unchanged body morphology, and no apparent differences in CNS apoptosis, proliferation, or patterning at embryonic stages. There was a reduction in expression of the known foxP2 target gene cntnap2 that was rescued by injection of wild-type foxP2 transcript. When we examined axon pathfinding using a pan-axonal marker or transgenic lines, including a foxP2-neuron-specific enhancer, we did not observe any axon guidance errors. Our findings suggest that foxP2 is not necessary for axon pathfinding during development.

Extraocular ectoderm triggers dorsal retinal fate during optic vesicle evagination in zebrafish

Dorsal retinal fate is established early in eye development, via expression of spatially restricted dorsal-specific transcription factors in the optic vesicle; yet the events leading to initiation of dorsal fate are not clear. We hypothesized that induction of dorsal fate would require an extraocular signal arising from a neighboring tissue to pattern the prospective dorsal retina, however no such signal has been identified. We used the zebrafish embryo to determine the source, timing, and identity of the dorsal retina-inducing signal. Extensive cell movements occur during zebrafish optic vesicle morphogenesis, however the location of prospective dorsal cells within the early optic vesicle and their spatial relationship to early dorsal markers is currently unknown. Our mRNA expression and fate mapping analyses demonstrate that the dorsolateral optic vesicle is the earliest region to express dorsal specific markers, and cells from this domain contribute to the dorsal retinal pole at 24 hpf. We show that three bmp genes marking dorsal retina at 25 hpf are also expressed extraocularly before retinal patterning begins. We identified gdf6a as a dorsal initiation signal acting from the extraocular non-neural ectoderm during optic vesicle evagination. We find that bmp2b is involved in dorsal retina initiation, acting upstream of gdf6a. Together, this work has identified the nature and source of extraocular signals required to pattern the dorsal retina.

Rapid and efficient zebrafish genotyping using PCR with high-resolution melt analysis.

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA). This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts. Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

Stereoacuity after small aperture corneal inlay implantation

Purpose The aim of this study was to compare stereoacuity before and after KAMRA corneal inlay implantation for the correction of presbyopia. Patients and methods This is a prospective study of 60 patients who underwent KAMRA inlay implantation. Patients were examined before and 6 months after surgery for stereoacuity, uncorrected distance visual acuity (UDVA), and uncorrected near visual acuity (UNVA). Results The mean stereoacuity before surgery was 29.5±28.1 arcsec (range: 20–200) and at 6 months was 29.8±26.4 arcsec (range: 20–200). The decline in stereoacuity was not statistically significant. At 6 months follow-up, UDVA was 20/25 or better in all 60 patients and UNVA was J2 (20/25) or better in 51 (85%) patients. Conclusion There is no significant change in stereoacuity following KAMRA inlay implantation. The KAMRA inlay is a good treatment option for improving near vision in presbyopic patients while preserving stereoacuity and distance vision.

Six-month visual outcomes for the correction of presbyopia using a small-aperture corneal inlay: single-site experience

Objective The objective of this study was to describe 6-month postoperative efficacy and safety outcomes after monocular KAMRA corneal inlay implantation in emmetropic presbyopic patients. Study design This study followed a retrospective chart analysis. Setting This study was performed at Hoopes Vision in Draper, UT, USA. Subjects and methods Fifty-seven patients met the inclusion criteria of this study and underwent KAMRA corneal inlay implantation following the approval of the United States Food and Drug Association between May 2015 and April 2016 at a single site. Surgery involved femtosecond laser-created corneal pockets of various depths. Efficacy, safety, and patient satisfaction reports were analyzed at 3 and 6 months. Results At 6 months follow-up, the monocular uncorrected near visual acuity (UNVA) was Jaeger (J) 4 (20/32), the mean uncorrected distance visual acuity was 20/25, and the mean corrected distance visual acuity was 20/20. At 6 months, 71% of patients with a pocket depth of ≥250 μm had a UNVA of 20/20 or better, whereas only 22% of patients with a shallow pocket depth of <250 μm had a UNVA of 20/20 or better. There was no statistical difference in UNVA at 6 months between virgin eyes and post-LASIK eyes. One patient had an explant and five patients underwent inlay recentration, all of which resulted in improved visual acuity. At 6 months, 72% of patients reported some level of satisfaction, 26% of patients reported being “not dependent” on reading glasses, and 62% of patients reported being able to do most things in bright light without reading glasses. Conclusion For patients with emmetropic presbyopia, the KAMRA inlay is a viable treatment option resulting in improved UNVA. Increased pocket depth may be associated with better postoperative outcomes. Safety rates are high, while explantation and recentering rates are low. Overall, patient satisfaction of the KAMRA inlay is good.

Solitary Plasmacytoma of the Petrous Apex: A Case Report and Review of Literature.

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Rainbow glare after laser-assisted in situ keratomileusis: a review of literature

This article reviews the current literature pertaining to rainbow glare (RG), including incidence rate, clinical presentation, etiology, prognosis, and management. RG is a rare optical complication of femtosecond laser-assisted in situ keratomileusis that results in patients seeing an array of spectral bands surrounding point sources of light under mesopic and scotopic conditions. The mechanism is thought to be a consequence of the formation of a transmissive diffraction grating on the posterior surface of the corneal flap created by the FS laser. RG has a good prognosis and is usually self-limiting. Persistent RG with concomitant residual refractive error may warrant lifting the flap and photoablating the posterior surface of the flap. Patients with persistent RG and no residual refractive error should be considered candidates for phototherapeutic keratectomy on the posterior flap surface.

Long-term changes in keratometry and refraction after small aperture corneal inlay implantation

Purpose To assess longitudinal refractive, keratometric, and topographic changes following KAMRA small-aperture inlay implantation. Design and setting Prospective study at a single site refractive surgery center. Methods Fifty patients underwent KAMRA small-aperture corneal inlay implantation for the correction of presbyopia. Uncorrected near visual acuity (UNVA), uncorrected distance visual acuity, manifest refractive spherical equivalent (MRSE), mean keratometry (Km), corneal topography, and surgically induced astigmatism vector analysis assessments were performed preoperatively and at 1, 3, 6, 12, 24, and 36 months postoperatively. Results The study comprises 50 eyes. An average shift of 0.15±0.63 D (range −1.63 to 2.00 D) occurred between preoperative baseline and 36 months. At 36 months, 54% of patients had hyperopic MRSE and 40% had myopic MRSE compared with baseline. Km was significantly elevated at all postoperative measurements compared with baseline, with the largest Km measured at 12 months. Eighty-six percent of patients had UNVA of 20/32 or better and 88% uncorrected distance visual acuity of 20/25 or better at 36 months. Longitudinal corneal topography revealed a pattern of corneal steepening over the body of the inlay and flattening over the aperture, correlating with a hyperopic shift. There was no significant surgically induced astigmatism. Conclusion KAMRA inlay may cause an increase in Km compared with baseline. Corneal steepening may occur in a specific pattern with steepening over the inlay and flattening over the aperture. This topographic pattern causes a hyperopic shift, which may be relevant for subsequent procedures, such as cataract extraction.